Coagulation of blood is a complex process requiring the sequential interaction of a large number of components, nearly all of which are proteins. These components include Fibrinogen, Factors II, V, VII, VIII, IX, X, XI, and XII. A lack of any of these components, or a nonfunctional component, can lead to an inability of the blood to clot when required, with the resultant excessive and life-threatening blood loss to the patient.
Hemophilia A is a common bleeding disorder caused by a deficiency or abnormality of Factor VIII. The severity of this disorder clearly demonstrates the importance of Factor VIII in the pathway of blood coagulation, even though this protein is only found in trace amounts in normal plasma. Factor VIII is present in plasma as a high-molecular-weight complex (Factor VIII complex), which includes Factor VIII:C and von Willebrand factor (Factor VIII:R or vWf). Factor VIII:C promotes blood coagulation. Factor VIII:R interacts with platelets to promote aggregation of the platelets and, when incorporated in the Factor VIII complex, acts as a stabilizer for Factor VIII:C.
The primary therapeutic use of Factor VIII has been its intravenous administration to hemophilia A patients. In severe cases, relatively high concentrations of Factor VIII are required. These high concentrations are obtained by purification and concentration of Factor VIII. However, purification often leads to instability and loss of Factor VIII:C activity because of the removal of Factor VIII:R from the Factor VIII complex during purification. Thus, the resultant purified product is a mixture of both stable Factor VIII complex and unstable Factor VIII:C, along with contaminating proteins that have not been removed, and proteins, such as albumin, which have been added to the product to stabilize the Factor VIII:C. Since these solutions contain an undesirably large portion of contaminating and stabilizing proteins, and since only Factor VIII:C is effective in treating hemophilia A patients, larger amounts of proteins have to be infused into patients than would be required if all the protein were Factor VIII:C.
Affinity chromatography has frequently been used as a method for separating a desired protein from other contaminating proteins. This method relies on a specific interaction between the desired protein and a ligand attached to the chromatographic medium. The specificity of the interaction in affinity chromatography is conferred by the unique ability of a given protein to recognize and bind to specific chemical compounds which are used as the ligands. The tremendous advantage of this method of chromatography over other methods commonly used (e.g., ion-exchange), is that only the proteins which recognize the ligand will bind to the column; thus, impurities and inactive protein may be removed from the desired protein. Elution of the specifically bound protein can be achieved in an ion-exchange manner by using solutions containing high salt or, more selectively, in an affinity manner by using a second chemical compound that is recognized by the protein. When the solution containing the second chemical compound is added, the bound protein will dissociate from the ligand and bind to the "mobile" second chemical compound in the solution and elute from the column.
Ion-exchange chromatography differs from affinity chromatography in that it relies only on ionic interactions between the protein and the chromatographic medium. Such ionic interactions are non-specific. In this case, the ligands bound to the chromatography medium contain charged groups, either negatively or positively charged, rather than a chemical compound that is specifically recognized by the protein. Elution from the ion-exchange medium is also non-specific and relies only on high salt concentrations. In ion-exchange chromatography, the salt competes for the charged groups of the chromatographic medium and the protein, resulting in elution of the protein from the chromatographic medium.
Another method that has been used for the purification of Factor VIII is the binding of the Factor VIII:R portion of the Factor VIII complex to monoclonal antibodies and the subsequent elution of the Factor VIII:C from the Factor VIII-Antibody complex. This procedure results in Factor VIII:C of very high specific activities, approximately 1500-2500 units/mg of total protein, and, therefore, high purity. (The phrase "specific activity" as used herein means units of Factor VIII:C clotting activity per milligram of protein. A "unit" is defined as the amount of Factor VIII:C in one ml of normal plasma.) However, the Factor VIII:R is dissociated from Factor VIII:C during this procedure, which results in the Factor VIII:C being unstable. To overcome the instability of Factor VIII:C, large amounts of stabilizing proteins, such as human serum albumin, are added to the purified Factor VIII:C. As has been the drawback with other purification methods (discussed above), the addition of stabilizing proteins is required, and larger amounts of proteins have to be infused into patients than would be required if all the protein were Factor VIII:C.
Currently, the methods used to purify Factor VIII result in protein preparations, i.e., Factor VIII concentrates, that have a relatively low Factor VIII specific activity due to contaminating and stabilizing proteins. When the Factor VIII concentrate has a relatively low Factor VIII:C specific activity, patients must be infused with undesirably large amounts of the extraneous protein to obtain the necessary level of Factor VIII required for effective treatment. The amount of protein needed to be infused could be drastically reduced if Factor VIII complex were the only protein contained in the infusion solutions. It is therefore desirable that there be provided an improved process for the separation of Factor VIII complex, i.e., the intact Factor VIII:C/Factor VIII:R complex, from contaminating proteins.